The present invention is directed to chirally enriched synthetic Oligomers which are chirally pure chirally enriched for phosphonate linkages of a preselected chirality and to methods for their synthesis for chirally enriched oligomers. In particular, we have found that chirally enriched Oligomers enriched for Rp methylphosphonate internucleosidyl linkages have enhanced binding affinities for RNA as compared to racemic all methylphosphonate internucleosidyl linkages. The chirally enriched Oligomers of the present invention may be enriched for methylphosphonate internucleosidyl linkages of Rp chirality, or for methylphosphonothioate internucleosidyl linkages of either Rp or Sp chirality.
Oligomers having naturally occurring phosphodiester internucleosidyl linkages and certain other internucleosidyl linkages do not have chiral centers at the phosphorus atom (or other atom) of the internucleosidyl linkage.
However, these phosphonate internucleosidyl linkages which include methylphosphonate and methylphosphonothioate internucleosidyl linkages are chiral at the phosphorus and have either Rp or Sp chirality depending on the relative orientation of the hydrogen or alkyl group. Thus, Oligomers having such internucleosidyl linkages may theoretically have 2.sup.n different diastereomeric forms for a particular Oligomer where n is the total number of phosphonate and internucleosidyl linkages in the Oligomer sequence. For example, an 11-mer having 10 phosphonate internucleosidyl linkages theoretically would have 1,024 diastereoisomers and a 19-mer having 18 phosphonate internucleosidyl linkages theoretically would have 262,144 diastereoisomers.
The reported effects of chirality of internucleosidyl linkages on the resulting oligomers and their biological or physical chemical behavior have been varied.
The preparation of two isomers of decathymidylate analogues having stereoregular, alternating methylphosphonate and phosphodiester backbones has been reported (Miller, et al., J. Biol. Chem. 255(20):9659-9665 (1980). Complexes between oligomers containing the two isomers and complementary polynucleotides were studied. The absolute configurations of the methylphosphonate groups of isomers 1 and 2 were not determined. Complexes formed by oligomers containing the two isomers with complementary polynucleotides were said to have different stoichiometries and thermal stabilities. Miller et al. hypothesized that in formation of a complex with a decathymidylate analog whose methylphosphonate groups were in the Sp-configuration the methyl group should have the least perturbational effect on solvent interactions with the complex; whereas, in contrast, complex formation with the decathymidylate analog whose methylphosphonate groups have the Rp-configuration would orientate the methyl group away from the base stacking region and toward the solvent and should result in "unfavorable interactions between the exposed methyl groups and the surrounding solvent."
Studies on complexes of duplex formation between a 19-mer phosphodiester oligonucleotide (dA.sub.19, dU.sub.19 or dT.sub.19) and a 19-mer methylphosphonate oligonucleotide having one phosphodiester 5'-internucleosidyl linkage (dA*.sub.19, dU*.sub.19 or dT*.sub.19) reported that transition curves for complexes between da*.sub.19 and dT.sub.19 or dU.sub.19 were sharp and similar to those for dA.sub.19 and dT.sub.19 or dU.sub.19, whereas transition curves for duplexes of dT*.sub.19 or dU*.sub.19 and dA.sub.19 were significantly broader, suggesting to the authors that methylphosphonate chirality had a significant influence on binding stability only when the pyrimidine strand was substituted. (Kibler-Herzog, Laura, et al., Nucleic Acids Research 18(12):3545-3555 (1990)).
A study of 2-diastereoisomeric pairs of octathymidine methylphosphonates (all Sp and SpSpSpRpSpSpSp versus all Rp and RpRpRpSpRpRpRp) compared with octathymidylic acid and a random mixture of octathymidine methylphosphonate diastereoisomers and complexes formed with penta-decadeoxyriboadenylic acid reported that configuration of the internucleosidyl methylphosphonate linkages may affect binding of (dA).sub.15 to the Oligomer and that the methyl in the Sp configuration decreased duplex stability. (Lesnikowski et al., Nucleic Acids Research 18(8):2109-2115 (1990).
Certain computer modeling studies reported that methylphosphonate ("MP") hybridization to a DNA target was predicted to be more stable with MP(Rp) substitution due to favorable hydrophobic interactions whereas MP(Sp) destabilized the double helix with less favorable hydrophobic interactions. The simulations compared antisense Oligomers having a single MP(Rp) to MP(Sp) substitution. (Hausheer et al., J. Am. Chem. Soc. 114:3201-3206 (1992)).
Computer modeling studies to determine the relative stability of Rp and Sp methylphosphonate Oligomers by free-energy perturbation approaches using a free-energy decomposition method were reported. The study reported that in the case of the Sp diastereomer the C2' and C3' sugar (5' direction) carbons and hydrogens unfavorably interacted with the methyl group, while the C5' sugar (3' direction) hydrogens destabilized the Rp diastereoisomer. Although the study reported the stability of the Rp-configuration to be favored, it was noted that under certain circumstances there may be reversals in stability of Rp and Sp diastereoisomers. (Ferguson and Kollman, Antisense Research and Development, 1:243-25 (1991)).
Studies of formation of duplexes using self-complementary DNA Oligomers having one methylphosphonate internucleosidyl linkage were reported. With the Rp duplexes, reported Tm increased when the substitution was closer to the 3'-end of the strand. With the Sp duplexes, substitution nearer the center of the strand was said to produce larger effects (e.g., greater Tm depressions) than substitution closer the either end of the duplex. In one instance of substitution between the second and third nucleoside (from the 5'-end) the Sp duplex had a higher Tm than the corresponding Rp duplex. (Bower et al., Nucleic Acids Research 15(12):4915-4930 (1987)).
In a summary of molecular modeling studies on single stranded, as well as base paired, forms of dinucleoside methylphosphonates it was reported that neither MP(Sp) nor MP(Rp) seemed to significantly alter the stereochemistry of duplex structure (Latha et al., J. Biomolecular Structure Dynamics, 9(3):613-631 (1991).
In a review article summarizing certain work on antisense agents, disadvantages of poorly hybridizing racemic oligodeoxynucleoside methylphosphonates in cell free extracts were said to be more or less balanced by their proposed advantages* in cell culture systems. It was noted that certain reports using a normal (deoxyribonucleoside) octamer with one methylphosphonate linkage found the oligomer with an Rp bond to have a melting temperature higher than the Oligomer with an Sp bond. It was also noted that sequence dependence of methylphosphonate base pairing might be as important as chirality. (Wickstrom, "Antisense DNA Therapeutics Neutral Analogs and Their Stereo-chemistry" in Gene Regulation: Biology of Antisense RNA and DNA, 119 to 132 (Erickson and Izant, eds., Raven Press Ltd., New York (1992)) FNT *Greater longevity, more efficient cellular uptake and lack of charge.
Diastereoselective synthesis of dinucleoside methylphosphonates containing thymidine has been reported (Engels et al., Nucleosides & Nucleotides 10 (1-3):347-350 (1991)). Diastereoselective synthesis of certain other dinucleoside methylphosphonates using methyldichlorophosphine has been reported (Loschner et al., Tetrahedron Letters 30(41):5587-5590 (1989)).